Method and vector for expression and isolation of biologically active molecules in urine of transgenic animals

ABSTRACT

A vector is provided which contains a promoter construct linked to a heterologous gene encoding a selected biologically active molecule or oncogene wherein the promoter construct is capable of directing urothelial expression of the heterologous gene. Methods of isolating biologically active molecules from urine of animals transfected with this vector and transgenic animals containing this vector are also provided.

BACKGROUND OF THE INVENTION

[0001] Methods of producing biologically active molecules by transfer ofrecombinant genes into cell in culture and into live animals have beendeveloped. For example, DNA molecules have been introduced into culturedcells by calcium phosphate precipitation or electroporations. Graham andVan der Ebb Virology 1973, 52, 456-467; Perucho et al. Cell 1980, 22,9-17; Chu et al. Nucleic Acids Research 1987, 15, 1311-1326; and Bishopand Smith Molecular Biology Medicine 1989, 6, 283-298. DNA moleculeshave also been introduced into the nucleus of cells in culture by directmicroinjection. Gordon et al. Proc. Natl Acad. Sci. USA 1980, 77,7380-7384; Gordon and Ruttel Methods in Enzymology 1983, 101, 411-433;and U.S. Pat. No. 4,873,191.

[0002] However, there are two major problems of producing biologicallyactive molecules such as protein products on a commercially viable scalevia these methods. First, bacterial expression systems frequently failto modify the proteins properly, i.e., by glycosylation, etc. Second,the subsequent isolation of gene products from the expression systemscan be extremely difficult. In bacteria, yeast, and baculovirus systemsthe expressed proteins are most often purified from insolubleintracellular compartments. Secreted proteins in yeast requirespecialized protease-deficient strains coupled with appropriate vectorswith secretion signals.

[0003] Retroviral vectors have also been used to introduce DNA moleculesinto the genome of animals. Jaenisch et al. Cell 1981, 24, 519; Sorianoet al. Science 1986, 234, 1409-1413; and Stewart et al. Embo. J. 1987,6, 383-388. Recombinant genes have been introduced into primary culturesof bone marrow, skin, fibroblasts, or hepatic or pancreatic cells andthen transplanted into live animals. There has also been success inusing mammary gland-specific promoters to drive the expression offoreign proteins in these secretory glands, ultimately leading to theirsecretion in the resultant milk. This method has been used commerciallyto express human growth hormone in cows and sheep. WO 94/05782. Thecopious volumes of milk produced by cows and sheep make this procedureattractive. However, this method suffers from several potentialdrawbacks: one being that the expressed protein even at relatively highlevels must be purified away from a large amount of milk proteins suchas caseins, immunoglobins, lactoferrins which may also entrap thedesired valuable product; another being that certain protein productsmay be insoluble in the calcium-rich environment of milk fluid; andanother being that this method requires the use of pregnant animalswhich are expensive and time consuming to produce.

SUMMARY OF THE INVENTION

[0004] An object of the present invention is to provide a vectorcomprising a promoter construct capable of directing urothelial geneexpression of a heterologous gene encoding a selected biologicallyactive molecule linked thereto. The vectors of the present invention areuseful in directing the expression of the heterologous gene inurothelial cells transfected with the vector which then secrete theencoded gene product into the urine for isolation, thus transforming thebladder into a bioreactor.

[0005] Accordingly, another object of the present invention is toprovide a method of producing a selected biologically active molecule inurine of an animal wherein urothelial cells in the animal aretransfected with the vector so that the heterologous gene of the DNAsequence is expressed and the selected biologically active molecule isrecovered in urine produced by the animal.

[0006] Another object of the present invention is to provide nonhumantransgenic animals produced using this vector.

[0007] Another object of the present invention is to provide animalmodels for human bladder cancer.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIGS. 1a and 1 b show the organization and nucleotide sequence ofthe mouse uroplakin II (UPII) genomic DNA. FIG. 1a provides theexon-intron organization of mouse UPII gene. The open and filled thickboxes denote the five coding sequences (exons) and non-coding sequences(introns), respectively, of the gene. The open and filled thin boxesrepresent a (CA)_(n) dinucleotide repeat region and an Alu-like murineB1 repeat, respectively. G1 and G2 designate two independent andpartially overlapping genomic clones. The restriction sites are SacI(S), NcoI (N), BamHI (B), SalI (Sal), and XhoI (X). FIGS. 1b-1 and 1 b-2provide the nucleotide sequence (SEQ ID NO:1) of a 4-kb SacI fragment ofmouse UPII gene. A reversed El repetitive sequence (in the 5′ upstreamregion) and a potential polyadenylation site (AATAAA; in the 3′untranslated region) are underlined and double-underlined, respectively.The wavy arrow denotes the transcriptional initiation site. Brokenarrows marked 1 to 4 denote the intron/exon junctions of the fourintrons. The predicted first amino acid residue of mature UPII proteinsequence is marked with an asterisk. The preceding domain contains a preand a pro sequence of 25 and 59 amino acids, respectively.

[0009]FIG. 2 illustrates the tissue distribution of UPII mRNA as assayedby RT-PCR. Poly(A)+mRNAs (0.3-0.4 mg) from mouse bladder (lanes 1 and13), skin (2), forestomach (3), glandular stomach (4), kidney (withoutrenal pelvis) (5), liver (6), spleen (7), testis (8), andthalamus/hypothalamus (9), cerebral cortex (10), and cerebellum (11)regions of the brain were reverse-transcribed, and amplified with eitherUPII-specific primers (Upper; 266 bp) or glyceraldehyde-3-phosphatedehydrogenase (GDH)-specific primers (Lower, as an internal control forcomparison; 130 bp). The PCR products were then electrophoresed on a1.3% agarose gel and stained with ethidium bromide. Lane 12 is anegative control (no cDNA template). The 266-bp UPII product wasdetected in abundance in bladder, but not in any other tested tissues,including the hypothalamus.

[0010]FIGS. 3a and 3 b illustrate the construction and quantitation of arepresentative transgene. FIG. 3a provides a restriction map(abbreviations as described in FIG. 1) of the endogenous murine UPIIgene. A 500-bp PCR fragment (thick bar) was used as a probe whichdetects a 1.4-kb NcoI fragment of the endogenous UPII genome but ashorter 1.1-kb NcoI fragment of the transgene. FIG. 3b provides arestriction map of the transgene. A 3.6-kb 5′-flanking sequence of theUPII gene was inserted into an Escherichia coli β-galactosidase(β-gal)-encoding placF vector. In this particular test expressionvector, a sequence containing a part of exon 1 and all of intron 1 andexon 2 of the mouse protamine-1 gene (mp1) was placed at the 3′-end ofthe β-gal (or lacZ) gene to provide an exon/intron splicing site and apolyadenylation signal. This chimeric gene was cut out from the vector,gel-purified, and microinjected into mouse eggs.

DETAILED DESCRIPTION OF THE INVENTION

[0011] Urine in the bladder is of relatively high osmolality (50 to1,000 mosmol/kg), with pH values as low as 4.5 and high concentrationsof urea and ammonium. The lumen of the bladder therefore provides anadvantageous environment for the production of proteins that arenormally difficult to express due to insolubility. The urea and highosmolality may serve as in situ denaturants and chaotropic agents.However, urine contains relatively little protein, in comparison withmilk, as the kidneys are designed to prevent protein loss, thereforeurothelial promoter-driven expression of proteins which by-passes thekidney produces the desired protein in a solution with relatively littlecontaminating host endogenous proteins.

[0012] In the present invention, a vector and method have been developedfor expressing biologically active molecules in the luminal cavity ofthe bladder of transgenic mammals.

[0013] The vector of the present invention comprises a promoterconstruct capable of directing urothelial gene expression of aheterologous gene encoding a selected biologically active moleculelinked to the promoter construct. Promoters active in directingexpression in the urothelium can be routinely identified in accordancewith the teachings provided herein and used in the promoter construct ofthe vectors of the present invention.

[0014] Urothelium, also known as transitional epithelium, is amultilayered epithelium that covers the surface of much of theurogenital tract including the renal pelvis, ureter, the entire bladderand a portion of the urethra. The apical surface of urothelium, indirect contact with the urine, is covered with numerous rigid lookingplaques. These plaques cover a large portion of the apical surface ofmammalian urothelium. Hicks, R. M. J. Cell Biol. 1965, 26, 25-48; Koss,L. G. Lab. Invest. 1969, 21, 154-168; Staehelin, L. A. J. Cell Biol.1972, 53, 73-91. They are believed to play a crucial role as apermeability barrier (Hicks, R. M. Biol. Rev. 1975, 50, 215-246) and/oras physical stabilizer of the urothelial cell surface (Staehelin, L. A.J. Cell Biol. 1972, 53, 73-91). When viewed in cross section, the outerleaflet of the plaque is almost twice as thick as the inner one, hencethe term “asymmetrical unit membrane” or “AUM” has been used to describethese plaques.

[0015] It has recently been shown that AUM contain 4 major integralmembrane proteins which are called uroplakin Ia (UPIa; 28 kDa),uroplakin Ib (UPIb; ˜27 kDa), uroplakin II (UPII; 15 kDa) and uroplakinIII (UPIII; 47 kDa). EM-immunolocalization studies established thatthese uroplakins are AUM-associated in situ, thus establishing them asthe major protein subunits of urothelial plaques. Yu et al. J. CellBiol. 1990, 111, 1207-1216; Wu et al. J. Biol. Chem. 1990, 265,19170-19179. Immunohistochemical survey of various bovine tissuesestablished that these UPs are urothelium-specific being present in theupper cell layers of the urothelia that cover the urogenital tractincluding the renal pelvis, ureter, bladder and part of the urethra.These data established uroplakins as excellent markers for an advancedstage of urothelia differentiation. Yu et al. J. Cell Biol. 1990, 111,1207-1216; Wu et al. J. Biol. Chem. 1990, 265, 19170-19179. Furthermore,uroplakins Ia, Ib, II and III appear to be the major protein componentsof all mammalian urothelial plaques. They are found in eight othermammalian species (human, monkey, sheep, pig, dog, rabbit, rat, andmouse), and the AUMs of these species appear morphologically similar,bearing crystalline patches of 12-nm protein particles with acenter-to-center spacing of 16.5 nm. Wu et al. J. Biol. Chem. 1994, 269,13716-13724.

[0016] The primary structures of UPs have recently been elucidated bycDNA cloning. The results established the existence of two closelyrelated UPI isoforms, the 27-kDa UPIa and the 28-kDa UPIb. Yu, J. J.Cell Biol. 1994, 125, 171-182. The mRNAs of all four known UPs haverecently been shown to be urothelium-specific, indicating thatexpression of UP genes is transcriptionally regulated. Yu, J. J. CellBiol. 1994, 125, 171-182; Lin et al. J. Biol. Chem. 1994, 269,1775-1784; Wu, X.-R. and Sun, T.-T. J. Cell Sci. 1993, 106, 31-43.

[0017] The expression of the mouse UPII gene, like its bovinecounterpart, is urothelium- and late-differentiation stage-specific.Using transgenic mouse techniques, a 3.6-kb 5′ flanking region has nowbeen identified as a promoter comprising the cis-elements for directingthe expression of a heterologous reporter gene specifically andefficiently to the suprabasal cell layers of the urothelium in a mannersimilar to the endogenous UPII gene. Using this promoter, it has nowbeen found that foreign proteins can be directed to the upper celllayers of the bladder urothelium for expression and secretion intourine.

[0018] Using a bovine UPII cDNA as a probe, a 16-kb mouse genomic clone(G1) was isolated which contains an ˜2.5-kb transcribed region that isflanked by ˜3.5-kb and ˜10 kb of 540 -and 3′-sequences, respectively(see FIG. 1a). Alignment of the coding sequence with the UPII cDNAsequences of cattle (Lin et al. J. Biol. Chem. 1994, 269, 1775-1784),which are highly homologous, defined the exon/intron junctions of fourintrons (FIG. 1b). 5′-RACE (Frohman et al. Proc. Nat'l Acad. Sci. USA1988, 85, 8998-9002) experiments using mouse bladder mucosal mRNA as atemplate established that the transcription site of the UPII gene islocated at 60-bp 5′-upstream of the translation initiation codon and27-bp downstream of a putative TATA box. The 5′-upstream region containsan Alu-like B1 repetitive sequence (−830 bp) and a (CA)_(n) stretch(˜−2.1 kb) Finally, a polyadenylation signal resides ˜230 bp downstreamof the translation stop codon (see FIG. 1b).

[0019] The mouse UPII gene is also expressed in the urothelium. mRNAswere prepared from various mouse tissues and probed for the presence ofUPII sequences by reverse transcription-polymerase chain reaction(RT-PCR) assay. A large amount of UPII product of expected size (266-bp)was generated from the bladder, but not from skin, forestomach,glandular stomach, kidney, liver, spleen, testis, or thehypothalamus/thalamus cortex and cerebellum of the brain (see FIG. 2).

[0020] A rabbit antiserum previously prepared against a syntheticpeptide corresponding to the N-terminal amino acid sequence ELVSVVDSGSG(1-11) (SEQ ID NO:2) of mature bovine UPII (Lin et al. J. Biol. Chem.1994, 269, 1775-1784) immunohistochemically stains the 15-kDa bovineUPII and localizes it to the superficial cell layers of bovineurothelium. This antiserum cross-reacted well with mouse UPII, whichcontains an identical epitope, but migrates slightly slower at anapparent 17 kDa mass. Immunofluorescent staining of frozen sections ofmouse bladders showed that the UPII was associated with the all thesuprabasal cell layers, suggesting that the onset of UPII geneexpression in mouse was earlier than that in cattle.

[0021] To define the cis promoter elements for urothelial-specificexpression and to demonstrate that heterologous genes can be targeted tothe suprabasal urothelial cells as endogenous UPII, a transgenic mousewas constructed that contains a chimeric gene in which a lacZ reportergene was under the regulation of a 3.6-kb 5′-flanking sequence of themouse UPII gene (FIG. 3b). The DNA construct was injected intofertilized mouse eggs for transgenic mouse production. Southern blotanalyses of the tail DNAs showed that the transgene was integrated intothe genomes of 4 of 25 mice. Three of these animals transmitted thereporter gene into their progeny. Southern blot analyses establishedthat the genomic DNAs of these three transgenic lines, TG1, TG2, andTG3, contained roughly 40, 6, and 30 copies, respectively, of thereporter gene per diploid genome. Probing the same Southern blot withthe lacZ sequence showed that the transgenes of all three lines were intandem repeats and were integrated into independent sites.

[0022] In all three mice lines, the transgene was expressed in thesuprabasal cells of the bladder epithelium in an expression patternsimilar to the endogenous UPII gene. The staining correlated somewhatwith gene dosage, as it was intense in TG1 (40 copies) but moderate inTG2 (6 copies) and TG3 (30 copies). β-galactosidase activity was onlyobserved in the bladder and other urothelia of mice that had inheritedthe transgene, confirming that the activity was transgene-specific. Inall three transgenic mice, no β-galactosidase activity was detected inany of the non-urothelial stratified epithelia tested, including thoseof the skin, tongue, cornea, esophagus, and forestomach. The reportergene product was also undetectable in all other epithelia tested,including those of liver, lung, glandular stomach, small and largeintestine, uterus, and testis; or mesenchymal tissues, includingfibroblasts, endothelial cells, spleen, and various muscle cells.

[0023] Experiments have also been performed wherein uroplakin IIpromoter was used to drive the expression of the biologically activehuman growth hormone gene in the urothelium of transgenic mice. In theseexperiments, a vector was constructed with the 3.6-kb UPII promoterplaced upstream from a human growth hormone cDNA. The vector was theninjected into the fertilized mouse eggs for transgenic mouse production.Thirteen founder mice were generated. Of these, six (5 male and 1female) transmitted the transgene to their offspring. Immunofluorescencestaining of the bladder epithelium of these transgenic mice usingantibodies to hGH showed strong staining indicating high level ofexpression. Immunolocalization performed by high resolution electronmicroscopy showed the accumulation of electron dense, aggregates of hGHthat are labeled by immuno-gold particles conjugated with antibodies tohGH. Most of the hGH particles are found in the vesicles lined with theasymmetrical unit membrane that are normally involved in transportingthe uroplakins to the apical surface of the bladder epithelium. Inaddition, some of the hGH particles formed another distinct populationof cytoplasmic vesicles thus revealing the presence of a previouslyunrecognized secretory pathway that may normally operate at a low levelin bladder epithelium. The high level of overexpression of hGH makesthese vesicles easily visible.

[0024] Urine from these mice was collected and hormone levels determinedby radioimmunoassay. Many of the F1 offspring had a significant levelsof the human growth hormone in their urine (up to 300 ng/ml) thusdemonstrating that the biologically active molecule was secreted intothe urine. Further, blood concentrations of the hormone were less than 5ng/ml indicating that the synthesized hormone is secreted vectoriallyinto the bladder cavity rather than into the bloodstream.

[0025] Other urothelia closely related to the epithelium of the bladderknown to cover other areas of the urinary tract, such as the renalpelvis of the kidney, the ureter, and the urethra and which alsoelaborate AUM plaques, exhibit similar expression of the transgene.

[0026] These data show that a promoter active in directing expression inthe urothelium of an animal, such as the 3.6-kb 5′-flanking sequence ofthe mouse UPII gene, can drive both a heterologous reporter gene and agene for a biologically active molecule to express in the upper celllayers of the bladder epithelium. The lack of expression innon-urothelial tissues indicates a high degree of tissue-specificity anddemonstrates that the cis elements of this promoter region provide verytight regulatory control on tissue-specific anddifferentiation-dependent expression of a gene placed downstream of thepromoter. As these results were corroborated in independent transgeniclines with differing sites of transgene integration, they show that theinherent promoter activity is responsible for the tissue-specificexpression and is not due to the effect of neighboring sequences of thetransgene integration sites. This tight regulation is a very desirableproperty of any promoter used for production of foreign protein productsin host transgenic animals, as it assures correct delivery to targetproduction sites, high efficiency of expression of transduced genes, andminimizes toxic effect of aberrant expression.

[0027] It has also been found that a urothelium specific promoter candirect expression of a human oncogene to the urothelium and is useful inthe production of animal models for human bladder cancer. In theseexperiments, a chimeric gene comprising a 3.6 kb 5′-flanking sequence ofmouse uroplakin II gene and a 2.8 kb SV40 large T oncogene wasconstructed. This chimeric gene was then microinjected into fertilizedmouse eggs which were implanted into foster mothers to producetransgenic mice expressing the oncogene. Four transgenic founder micecarrying uroplakin II/SV40 large T chimeric genes were identified fromthirty live births. Two of these positive founder mice harboring 10 and6 copies of transgenes succumbed with bladder tumors at ages of 3 and 5months, respectively. Histological examination revealed tumors wereinvasive transitional cell carcinomas, resembling those occurring inhumans. Reverse transcriptase polymerase chain reaction confirmed theexpression of SV40 large T oncogene in urothelial tumors, but not innon-urothelial, normal tissues. Immunohistochemical staining showed thetypical nuclear staining of SV40 large T antigen in bladder tumor cells.The remaining two transgenic mice carried lower copy number oftransgenes and exhibited a urothelial morphology resembling carcinoma insitu as is seen in humans. In contrast, no tumors were seen in over 50transgene negative mice.

[0028] While all of the experiments discussed above were conducted usingthe mouse UPII promoter, as will be obvious to those of skill in the artupon this disclosure, other promoter constructs capable of directingurothelial gene expression can used to yield similar results. Forexample, mouse uroplakin II 5′-upstream sequences that are shorter orlonger than 3.6-kb but can still achieve the same degree of urotheliumexpression. Also useful are DNA sequences with relatively minormodifications to the mouse UPII promoter, such as sequences with pointmutations, partial deletions or chemical modifications.

[0029] In addition, sequences that are related to the 3.6 kb 5′ flankingsequence of the mouse uroplakin gene, including, but not limited to,promoter sequences of uroplakin-II-homologous genes of other mammalianspecies such as human, cattle, sheep, goat, rabbit and rat, can also beused. There is sufficient similarity between this gene in differentspecies, so that similar results with the UPII promoter sequence inother animals is expected. For example, the UP gene organization (Ryanet al. Mamm. Genome 1993, 4, 656-661), cDNA (Lin et al. J. Biol. Chem.1994, 269, 1775-1784) and protein sequences, tissue patterns ofexpression, and morphology of AUMs are strikingly similar between themouse and cow. The amino acid sequence of bovine and mouse UPII arehighly similar, sharing 84 of their 100 amino acid residues. Wu et al.J. Biol. Chem. 1994, 269, 13716-13724. In addition, although the onsetof expression of the UPII gene is different in these two species, UPIIis clearly differentiation-related in both cow and mouse urothelia.

[0030] Further, promoters of other genes that are active in directingexpression in the urothelium are known and can also be used in thevectors of the present invention. Examples include, but are not limitedto, the promoter of uroplakin la (Yu et al. J. Cell Biol. 1994, 125,171-182; Yu et al. J. Cell Biol. 1990, 111:1207-16), uroplakin Ib (Yu etal. J. Cell Biol. 1994, 125, 171-182; Yu et al. J. Cell Biol. 1990,111:1207-16), uroplakin III (Wu, X. R., and Sun, T. T. J. of CellScience 1993, 106, 31-43, and the urohingin gene (Yu et al. EpithelialCell Biol. 1992, 1, 4-12).

[0031] Identification of additional promoters active in directing geneexpression in the urothelium is performed routinely using thesubtraction library technique. Using this technique which eliminates thecDNAs that are shared by multiple tissues (Diatchenko et al. Proc. Nat'lAcad. Sci. 1996, 93, 6025-6030), a library highly enriched in bladderspecific cDNAs was generated. Total RNAs were isolated from bovinebladder, kidney, lung, spleen, muscle, esophagus, stomach, intestine,colon, liver and brain. Northern blot analysis of these mRNAs using anactin cDNA as a probe demonstrated the intactness of the actin mRNA inall of these preparations. Bladder cDNAs were then used as the “tester”,and the cDNAs of all the other non-bladder tissues, referred to as the“drivers” were subtracted from the bladder cDNAs. The cDNAs of thenon-subtracted and the subtracted were probed using actin cDNA oruroplakin Ib. The results indicate that the original bovine bladder cDNApreparation contained abundant actin mRNA and relatively littleuroplakin Ib mRNA. In contrast, the subtracted library contained almostno detectable actin mRNA (at least 50 fold reduction) but greatlyincreased uroplakin Ib mRNA (>10 to 15 fold enrichment). Multiple cDNAclones have been isolated from the substraction library and used toprobe the mRNAs of various bovine tissues. For example, a uroplakin Ibprobe confirmed its bladder specificity. Tissue distribution patternshave also been determined for three unidentified partial cDNAs which arerelatively bladder specific. Sequencing data indicate these three clonesare novel genes not described previously. It is believed that thepromoters of these genes will also be useful in directing expression ofa heterologous gene for a biologically active molecule in the urotheliumof transgenic animals.

[0032] The vectors of the present invention are thus useful intransforming the bladder into a bioreactor capable of producing abiologically active molecule in the urine for isolation. In oneembodiment, this vector is introduced into germ cells to produce atransgenic animal capable of expressing the biologically active moleculein its bladder. As used herein, “biologically active molecule” refers toa molecule capable of causing some effect within an animal, notnecessarily within the animal having the transgene. Examples of suchmolecules include, but are not limited to, adipokinin,adrenocorticotropin, blood clotting factors, chorionic gonadotropin,corticoliberin, corticotropin, cystic fibrosis transmembrane conductanceregulators, erythropoietin, folliberin, follitropin, glucagongonadoliberin, gonadotropin, human growth hormone, hypophysiotropichormone, insulin, lipotropin, luteinizinghormone-releasing hormone,luteotropin, melanotropin, parathormone, parotin, prolactin,prolactoliberin, prolactostatin, somatoliberin, somatotropin,thyrotropin, tissue-type plasminogen activator, and vasopressin. Ofcourse, as will be obvious to one of skill in the art, the above list isnot exhaustive. In addition, new genes for biologically active moleculesthat will function in the context of the present invention arecontinually being identified. The biologically active molecule can beisolated from the urine of these transgenic animals. Accordingly, thepresent invention provides a means for isolating large amounts ofbiologically active molecules from the urine of transgenic animals whichcan be used for a variety of different purposes.

[0033] The vectors of the present invention have also been demonstratedto useful in directing expression of human oncogenes to the urothelia inof transgenic animals. In this embodiment, the vector is introduced intogerm cells to produce a transgenic animal which serves a model for humanbladder cancer.

[0034] In another embodiment, the vector comprises a system which iswell received by the urothelial cells lining the lumen of the bladder.An example of a useful vector system is the Myogenic Vector System(Vector Therapeutics Inc. Houston Tex.). In this embodiment, theheterologous gene of the biologically active molecule linked to thepromoter construct capable of directing urothelial expression andcarried in the vector is introduced into the bladder of an animal invivo. Introduction of the vector can be carried by a number of differentmethods routine to those of skill in the art. For example, a vector ofthe present invention could be placed in direct contact with theurothelium via a rubber urethral catheter or Foley catheter. Vectors ofthe present invention can also be incorporated into liposomes andintroduced into the animal in that form. The transgene is absorbed intoone or more epithelial cells capable of expressing and secreting thebiologically active molecule into the urine collecting in the bladder.It may be preferred for some biologically active molecules to alsoengineer a signaling sequence into the vector to insure that themolecule is secreted from the apical surface into the lumen. Use ofsignaling sequences such as the glycophosphatidylinositol (GPI) linkagein anchoring molecules to a selected surface is well known in the art.The biologically active molecule is then voided from the lumen where itcan be collected and separated from other components in the urine.

[0035] The following nonlimiting examples are provided to furtherillustrate the present invention.

EXAMPLES Example 1 Characterization of the Mouse UPII Gene

[0036] A bovine UPII cDNA (Lin et al., J. Biol. Chem. 1994, 269,1775-1784) was used as a probe to screen a mouse EMBL3-SP6A/T7 genomiclibrary (Clontech Laboratories Inc., Palo Alto, Calif.). Two overlappingclones (G1 and G2) were isolated (FIG. 1a) and were sequenced by thedideoxynucleotide termination method. The transcriptional initiationsite was determined by sequencing three clones of 5′-RACE (rapidamplification of cDNA ends) products of mouse bladder cDNA.

Example 2 Expression of a Fusion Gene (UPII-lacZ) in Transgenic Mice

[0037] A 6-kb XhoI DNA fragment of the G1 genomic clone (FIG. 1a) wassubcloned in pGEM7Z and then restriction-cut to yield a 3.6-kb DNAfragment of G1 clone (extending from the XhoI site at −3.6 kb to theBamHI site at −42 bp relative to the transcription initiation site) andinserted into the SmaI site of a lacZ vector, placF, (Peschon et al.Proc. Natl. Acad. Sci. USA 1987, 84, 5316-5319; Mercer et al. Neuron1991, 71 703-716) to generate pUPII-LacZ (FIG. 3). The 7.1-kb fusiongene was excised using Kpn I and Hind III, gel-purified, andmicroinjected into fertilized mouse eggs (from F1 hybrids of C57BL/6J XDBA2), which were implanted into CD-1 foster mothers. The lacZ transgenewas identified by Southern blot analysis of tail DNA in accordance withmethods well known in the art. Positive founder animals wereback-crossed with (C57BL/6J X DBA2) F1 hybrids to generate semizygousanimals that were used for studying transgene expression.

Example 3 Production of a Mouse Model of Bladder Cancer

[0038] A chimeric gene was constructed using a 3.6 kb 5′- flankingsequence of mouse uroplakin II gene and a 2.8 kb SV40 large T oncogenein accordance with procedures described in Example 2. The resultingchimeric gene was microinjected into fertilized mouse eggs which werethen implanted into foster mothers to generate transgenic mouse lines.Histological examination of tumors formed in these animals was performedto ascertain tumor cell type and invasiveness. Further, expression ofSV40 large T oncogene in the tumors of these mice was confirmed byreverse transcriptase polymerase chain reaction.

1 2 3963 Nucleic Acid Single Linear No 1 GAGCTCAGGT CCTATCGAGTTCACCTAGCT GAGACACCCA CGCCCCTGCA 50 GCCACTTTGC AGTGACAAGC CTGAGTCTCAGGTTCTGCAT CTATAAAAAC 100 GAGTAGCCTT TCAGGAGGGC ATGCAGAGCC CCCTGGCCAGCGTCTAGAGG 150 AGAGGTGACT GAGTGGGGCC ATGTCACTCG TCCATGGCTG GAGAACCTCC200 ATCAGTCTCC CAGTTAGCCT GGGGCAGGAG AGAACCAGAG GAGCTGTGGC 250TGCTGATTGG ATGATTTACG TACCCAATCT GTTGTCCCAG GCATCGAACC 300 CCAGAGCGACCTGCACACAT GCCACCGCTG CCCCGCCCTC CACCTCCTCT 350 GCTCCTGGTT ACAGGATTGTTTTGTCTTGA AGGGTTTTGT TGTTGCTACT 400 TTTTGCTTTG TTTTTTCTTT TTTAACATAAGGTTTCTCTG TGTAGCCCTA 450 GCTGTCCTGG AACTCACTCT GTAGACCAGG CTGGCCTCAAACTCAGAAAT 500 CCACCTTCCT CCCAAGTGCT GGGATTAAAG GCATTCGCAC CATCGCCCAG550 CCCCCGGTCT TGTTTCCTAA GGTTTTCCTG CTTTACTCGC TACCCGTTGC 600ACAACCGCTT GCTGTCCAAG TCTGTTTGTA TCTACTCCAC CGCCCACTAG 650 CCTTGCTGGACTGGACCTAC GTTTACCTGG AAGCCTTCAC TAACTTCCCT 700 TGTCTCCACC TTCTGGAGAAATCTGAAGGC TCACACTGAT ACCCTCCGCT 750 TCTCCCAGAG TCGCAGTTTC TTAGGCCTCAGTTAAATACC AGAATTGGAT 800 CTCAGGCTCT GCTATCCCCA CCCTACCTAA CCAACCCCCTCCTCTCCCAT 850 CCTTACTAGC CAAAGCCCTT TCAACCCTTG GGGCTTTTCC TACACCTACA900 CACCAGGGCA ATTTTAGAAC TCATGGCTCT CCTAGAAAAC GCCTACCTCC 950TTGGAGACTG ACCCTCTACA GTCCAGGAGG CAGACACTCA GACAGAGGAA 1000 CTCTGTCCTTCAGTCGCGGG AGTTCCAGAA AGAGCCATAC TCCCCTGCAG 1050 AGCTAACTAA GCTGCCAGGACCCAGCCAGA GCATCCCCCT TTAGCCGAGG 1100 GCCAGCTCCC CAGAATGAAA AACCTGTCTGGGGCCCCTCC CTGAGGCTAC 1150 AGTCGCCAAG GGGCAAGTTG GACTGGATTC CCAGCAGCCCCTCCCACTCC 1200 GAGACAAAAT CAGCTACCCT GGGGCAGGCC TCATTGGCCC CAGGAAACCC1250 CAGCCTGTCA GCACCTGTTC CAGGATCCAG TCCCAGCGCA GTATGGCATC 1300CACACTGCCT GTCCAGACCT TGCCCCTGAT CCTGATTCTG CTGGCTGTCC 1350 TGGCTCCGGGGACTGCAGGT CTCTATTGCT GGTGGGTGCG AGGAGGGTTT 1400 CAGAGCGCTA GACAGGGAACATTGTCTCCC CAGGGCTCTC AAGGACAGGA 1450 ATGTTGGTCT AGCTGGTTGG GGTTGAGAGTTACTAGTGGT AGGAATCAGG 1500 TGACAAATTC CTGGGCTTCT TCCCAGATCC AGGAGTCAAGAAATTTGGGT 1550 AAGTGTCCAA GGTTTGTGTG AGTTGGGCGA GACTGGGGAC TGACTGGGTG1600 CCATGGTCTA GTTTGGGTCG GTAGGGCTAT CTGGCTCCCA ACAGCGCGGC 1650GTACCCACCA TCTGCAGATC AAGCCTGCCA TCTGGTGGTC AGATCCACAC 1700 GCTCCTCTTCTGTCTCTGCA CCCTTAGCAA TGACCACCCA CCCACCCCGC 1750 CAGCTCTGAG TTAAGAGGGGGCTAACTCCT GAGTTCCCTC TCGGCTCCCT 1800 AACAGACTTC AACATCTCAA GCCTCTCTGGTCTGCTGTCT CCGGCGCTAA 1850 CAGAAAGCCT GTTAATTGCC TTGCCCCCAT GTCACCTCACGGGAGGTAAT 1900 GCCACATTGA TGGTCCGGAG AGCCAACGAC AGCAAAGGTA GACCTCCCTT1950 GTACCCATTT ATTCTACTCG TCGTAACCCC TCTTAACGAT ACCCAAGAGC 2000TGCCCGTTCT ACAAGAGTGG ACGCTAGAAT CTGATCTTGC CTTTCACTCC 2050 TATTTCCCCTCAGTGGTTAA GTCAGACTTT GTGGTGCCTC CATGTCGCGG 2100 GCGCAGGGAG CTTGTGAGCGTGGTGGACAG TGGGTCTGGC TACACCGTCA 2150 CAAGGCTCAG CGCATATCAG GTGACAAACCTAACACCAGG AACCAAATAC 2200 TAGTAGGTAC CGATGGACAC CTGTGGAGGT GGGATGGCAAAAAAGGGAAG 2250 TGGAGGTCCC GTGAGGGTGG GGAAGTGCCG GGAAGCATGA GTTAGAGAGG2300 GCACAGCTAA AGGGTAGGAA ATGTGAACCT GGACCCCAGG AGGGCCCAGA 2350TGGGACACAT AGCTAGAAGG TGGAGGCTGG AACCCCTCCT CCCGAGTGCC 2400 AGATACGTACAACCTCTGCT TTCTCTCAAC TCCGCCTCTA AAGCATATCC 2450 TACCGAGTAC AGAAGGGGACGTCGACCGAG TCCAGTCCAG AGACTCCCAT 2500 GTCCACGCTT CCTCGTTAAG TAAAATGCCCGTCTCTCACA CTTCCCTAAG 2550 CTCCGACTTT TTTCTCCTAG AGCAAGTTAG CTAAACTGTTTCCCGAGTGC 2600 TCAGTCGCAC ACACACCCCC TCCCCAACCC CCCAGTATTT GGTATGGCCC2650 CTCCTGTCCT GTTCAATCAT CTCTGCACTA GAGGTTCCTT GTGCAGAGGG 2700ATGATGTCCT CCTTGGTGGC TCCTAAGTGT TGCTGTGAGG GGGGTCTATG 2750 TTTGCTTGACTGGTTGGCTG GATGACCAGT TGAACTGATG CTGGAGGCTA 2800 CTGGATGGCT GGGCTAATGCTGTGAACCAC AGGAGCTACC TAGGAACCCC 2850 TTCAACTCAC AGAGGTTCCC CCATCTTCTTCTGACAGGAA AAAACATGGA 2900 GTCTATTGGG TTAGGAATGG CCCGGACAGG AGGGATGGTGGTCATCACAG 2950 TGCTGCTGTC TGTGGCCATG TTCCTGTTGG TCGTGGGTCT TATTGTTGCC3000 CTGCACTGGG ATGCCCGCAA ATGAAAAGGG CTCTCCTGCA TCCCAGGCTC 3050CTCCAAGAAG TCCAGCCTGC CTCCTGCCAG GCTGTAGTCA CTGGCTTCTC 3100 AGTGGCTTTTCTTCCCTCTC CCCGCCCCCT CCTCGAGTCC ACTCCTGACA 3150 GTGCCCCCTC CCTGCTCCCTGTCTCACCTT GCAGCACTCC CTGCTAGCCC 3200 CACTGCAATC CTGCCAACAC TGATTTATCTCTTAACTGTA CTTAATTCTC 3250 ACAATAAAGG CTGACCCACG TAGTATGTCT CATCTCCGACCATGTCTATG 3300 TGAGTCACCC CTTTAGCTGG TCCCCTTATG CACATATCAA AACTACCAAT3350 GTCAAGGTCA CGTGCATGTC ATTTTCTCTA TCCCAAACCC CAAGGGTGAC 3400TTTTACCAGG AGGGAGGCAA GCAGAGGCAG AGATAATGAA GCCTCAAGCC 3450 CAGACTAGGGAAGCCCTCCA AGCCCCAGAC CTAGGGCTTG GGTTTTGCAT 3500 CCTGCACTCA GTAGATACCCAAGCAGGAGT CTAGTTGGGC AGGGGGTAGA 3550 AGCTGGATCA CCATGTGAGC CTGACTGGGAAGCTGACAGA ACTAGGGAAG 3600 AACTAGAGAA AACACAAACA GGGCAGGCCC TCCAGCCCTGGGTGAAGAAC 3650 ATGCTAAACG GTTCTAGACC CCTAGAGCCG AGGTGGACGG AAGCTCCTGG3700 AAGGGGGAGG GGGGGACACA ACATAGGTAA ACAGGCAGTG GCACCCTCGT 3750CCATTTTTAA AATATAGTTT TGTTCTATAA AAGTTTTATT TATTTATTTA 3800 TTTGCTTGTTTTTATTTGTT TGTTTGTTTT CCAGAGCTGA GGCAAAAACC 3850 CAGGACCTTG AGCTTGCTAGGCAAGTGCTC TACCACTGAG CTAAATCCCC 3900 AACCCCTGTT TTTGTTTTTT TGAAGCAGGGTTTCTCTGTG TAGCTCTGGC 3950 TGTCCTAGAG CTC 3963 11 Amino Acid Linear 2GLU LEU VAL SER VAL VAL ASP SER GLY SER GLY 1 5 10

What is claimed is:
 1. A method of producing a selected biologicallyactive molecule in the urine of a non-human animal, comprising the stepsof: generating a non-human transgenic animal having cells, which carry aheterologous gene encoding a selected biologically active molecule, saidheterologous gene being linked to an epithelial-specific promoter whichselectively directs the expression and secretion of the selectedbiologically active molecule into the urine, wherein the heterologousgene encodes a biologically active molecule; and recovering theexpressed and secreted biologically active molecule in the urineproduced by the non-human animal.
 2. A method of producing a selectedbiologically active molecule in the urine of a non-human animal,comprising the steps of: transforming epithelial cells capable ofsecretion directly into the urine, by-passing the filtering effect ofthe kidney, in a non-human animal, with a promoter linked to aheterologous gene encoding a biologically active molecule, therebydirecting expression and secretion of the biologically active moleculeinto the urine; and recovering the expressed and secreted biologicallyactive molecule in the urine produced by the non-human animal.